ETHNOPHARMACOLOGICAL RELEVANCE:Bufei Huoxue capsule (BHC) as a classic Chinese patent medicine formula, has the efficacy of tonifying the lungs and activating the blood. It has been extensively used in China for the treatment of chronic obstructive pulmonary disease (COPD) clinically. However, its mechanism is still unclear, which hampers the applications of BHC in treating COPD.AIM OF THE STUDY:The purpose of the present study was to demonstrate the protective efficacy and mechanism of BHC on COPD model rats by integrating serum metabolomics analysis and network pharmacology study.MATERIALS AND METHODS:A COPD rat model was established by cigarette fumigation combined with lipopolysaccharide (LPS) airway drip for 90 consecutive days. After oral administration for 30 days, the rats were placed in the body tracing box of the EMKA Small Animal Noninvasive Lung Function Test System to determine lung function related indexes. Histopathological alteration was observed by H&E staining and Masson staining. The serum levels of inflammatory cytokine, matrix metalloprotein 9, and laminin were determined by ELISA kits. Oxidative stress levels were tested by biochemical methods. UHPLC-Q-TOF/MS analysis of serum metabolomics and network pharmacology were performed to reveal the bioactive metabolites, key components and pathways for BHC treating COPD. WB and ELISA kits were used to verify the effects of BHC on key pathway.RESULTS:BHC could improve lung function, immunity, lung histopathological changes and collagen deposition in COPD model rats. It also could significantly reduce inflammatory response in vivo, regulate oxidative stress level, reduce laminin content, and regulate protease-antiprotease balance. Metabolomics analysis found 46 biomarkers of COPD, of which BHC significantly improved the levels of 23 differential metabolites including arachidonic acid, leukotriene B4 and prostaglandin E2. Combined with the results of network pharmacology, the components of BHC, such as calycosin, oxypaeoniflora, (S)-bavachin and neobavaisoflavone could play therapeutic roles through the arachidonic acid pathway. In addition, the results of WB and ELISA indicated that BHC could suppress the expressions of COX2 and 5-LOX in lung tissues and inhibit the generation of AA and its metabolites in serum samples. Regulation of arachidonic acid metabolic pathway may be the crucial mechanism for BHC treating COPD.CONCLUSIONS:In summary, the studies indicated that BHC exhibited the protective effect on COPD model rats by anti-inflammatory and anti-oxidative properties through arachidonic acid metabolism pathway. This study provided beneficial support for the applications of BHC in treating COPD.

目的 研究补肺活血胶囊(Bufei Huoxue Capsules,BHC)在大鼠血浆、胆汁、尿液、粪便中的原型成分及其代谢产物,归纳总结不同类型化合物的体内代谢规律.方法 大鼠igBHC后,收集血浆、胆汁、尿液和粪便样品并进行预处理,采用超高效液相色谱-飞行时间质谱(ultra-high performance liquid chromatography-time-of-flight mass spectrometry,UPLC-Q-TOF-MS)及质量亏损过滤技术,分别在正、负离子模式下对原型成分及代谢产物进行分析鉴定.结果 共检测到没食子酸、芍药苷、毛蕊异黄酮、新补骨脂异黄酮、(S)-异补骨脂二氢黄酮、补骨脂苷等原型成分及代谢产物67个,其中酚酸类成分10个、单萜类成分7个、黄酮类成分35个、香豆素类成分12个、皂苷类成分3个,主要代谢途径包括氧化、还原、甲基化、葡糖醛酸结合、硫酸结合等.结论 初步阐明了 BHC大鼠体内的代谢产物及其代谢转化特征,为基于体内过程揭示其药效物质基础奠定了基础,为其临床安全用药提供了科学依据.

Zingiber officinale and Panax ginseng, as well-known traditional Chinese medicines, have been used together to clinically treat ulcerative colitis with synergistic effects for thousands of years. However, their compatibility mechanism remains unclear. In this study, the shift of gut microbiome and fecal metabolic profiles were monitored by 16S rRNA sequencing technology and ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry analysis, respectively, which aimed to reveal the synergistic mechanism of Zingiber officinale and Panax ginseng on the amelioration of ulcerative colitis. The results showed that the relative abundance of beneficial bacteria (such as Muribaculaceae_norank, Lachnospiraceae NK4A136 group and Akkermansia) was significantly increased and the abundance of pathogenic bacteria (such as Bacteroides, Parabacteroides and Desulfovibrio) was markedly decreased after the intervention of Zingiber officinale-Panax ginseng herb pair. And a total of 16 differential metabolites related to ulcerative colitis were identified by the metabolomics analysis, which were majorly associated with the metabolic pathways, including arachidonic acid metabolism, tryptophan metabolism, and steroid biosynthesis. Based on these findings, it was suggested that the regulation of the gut microbiota-metabolite axis might be a potential target for the synergistic mechanism of Zingiber officinale-Panax ginseng herb pair in the treatment of ulcerative colitis. Furthermore, the integrated analysis of microbiome and metabolomics used in this study could also serve as a useful template for exploring the mechanism of other drugs.

目的 建立超高效液相色谱法(UHPLC)同时测定补肺活血胶囊(Bufei Huoxue Capsules,BHC)中12种指标成分没食子酸、氧化芍药苷、芍药内酯苷、补骨脂苷、芍药苷、异补骨脂苷、毛蕊异黄酮苷、芒柄花苷、毛蕊异黄酮、补骨脂素、异补骨脂素、苯甲酰芍药苷的含量.方法 采用AcquityUPLCBEHC18色谱柱(100 mm×2.1 mm,1.7 μm);流动相为乙腈-0.1％甲酸水溶液,梯度洗脱;体积流量0.4mL/min,柱温35℃,进样量4μL,检测波长246 nm.结果 BHC中12种指标成分在18 min内色谱峰分离良好,没食子酸、氧化芍药苷、芍药内酯苷、补骨脂苷、芍药苷、异补骨脂苷、毛蕊异黄酮苷、芒柄花苷、毛蕊异黄酮、补骨脂素、异补骨脂素和苯甲酰芍药苷分别在0.38～195.20(r=0.999 9)、0.17～21.44(r=0.999 6)、0.85～433.80(r=0.999 9)、0.68～345.60(r=0.999 9)、1.11～566.60(r=0.999 8)、0.47～241.40(r=0.999 9)、0.08～39.36(r=0.999 8)、0.05～26.16(r=0.999 9)、0.03～16.42(r=0.999 9)、0.25～129.10(r=0.999 8)、0.26～133.10(r=0.999 7)、0.09～47.28 μg/mL(r=0.999 9)线性关系良好;精密度、稳定性、重复性、加样回收率均符合分析要求.11批补肺活血胶囊中没食子酸、氧化芍药苷、芍药内酯苷、补骨脂苷、芍药苷、异补骨脂苷、毛蕊异黄酮苷、芒柄花苷、毛蕊异黄酮、补骨脂素、异补骨脂素和苯甲酰芍药苷平均质量分数分别为2.6242、0.222 4、16.409 9、7.459 5、18.9742、7.887 5、0.505 7、0.109 9、0.170 7、1.364 7、1.170 0、0.203 6 mg/g.结论 所建立的方法分析速度快、准确度高、重复性好,为BHC质量标准提升提供了方法支撑.

Hui Medicine ZhaLi NuSi Prescription (ZLNS) is described in "Hui Hui Prescription" and it has been used to treat cerebral infarction in Hui Region, China. In this study, a rapid and reliable ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) method was established and applied to simultaneously determine geniposidic acid, oxypaeoniflorin, hydroxysafflor yellow A, caffeic acid, magnoflorine, paeoniflorin, ferulic acid, β-ecdysterone, icariin, rhein and baohuoside I in rat plasma. The pharmacokinetic parameters of these components and the influences of essential oil (EO) on them were investigated in normal rats. The results showed that the pharmacokinetic parameters (AUC0-t , AUC0-∞ , t1/2 , tmax , cmax ) of the above compounds were significantly changed after co-administering with ZLNS EO. The AUC values of oxypaeoniflorin, paeoniflorin, ferulic acid and baohuoside I with EO were decreased significantly. This is the first report for comparative pharmacokinetic study of ZLNS bioactive components in normal rats, which may provide the basis for drug interaction study in vivo, and an insight for their clinical applications.

This study was designed to determine the metabolites of Yiqi Baoyuan Prescription(YQBYP) in rats. The ultra-high performance liquid chromatography coupled to time-of-flight mass spectrometry(UPLC-TOF-MS) and mass defect filter(MDF) were employed to analyze the metabolites of YQBYP in rat plasma, bile, urine and feces. Chromatographic separation was conducted on Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) under gradient elution with 0.1% formic acid aqueous solution(A)-acetonitrile(B), and the column temperature was 30 ℃. Electrospray ion(ESI) source was used under positive and negative ion modes, with capillary voltage of 3.0 kV and mass scanning range of m/z 100-1 000. In this experiment, 9 prototype components and 36 metabolites were identified in rat plasma, bile, urine and feces samples. The results showed that the main metabolic pathways of YQBYP in rats involved methylation, demethylation, oxidation, and other phase Ⅰ reactions as well as glucuronidation, sulfation, and other phase Ⅱ reactions. This study provided scientific basis for clarifying the therapeutic material basis of YQBYP and product development.

An ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS) method was established to investigate the pharmacokinetic behaviors of psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, psoralen, isopsoralen, methylnissolin, and neobavaisoflavone in rat plasma after oral administration of Bufei Huoxue Capsules. After SD rats were administered with Bufei Huoxue Capsules suspension by gavage, blood samples were collected from the inner canthus at different time points. After protein precipitation, plasma samples were separated on ACQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm). The mobile phase consisted of acetonitrile(A) and water(B) containing 0.1% formic acid in gradient elution. The positive and negative ions were measured simultaneously in the multi-reaction monitoring(MRM) mode. The pharmacokinetic parameters were calculated and fitted by DAS 3.2.8. Psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, psoralen, isopsoralen, methylnissolin, and neobavaisoflavone were detected in the rat plasma after drug administration, with AUC_(0-t) of(3 357±1 348),(3 555±1 696),(3.03±0.88),(2.21±0.33),(1 787±522),(2 295±539),(5.69±1.41) and(3.40±0.75) μg·L~(-1)·h, and T_(max) of(1.56±0.62),(1.40±0.70),(0.21±0.05),(0.25±0.12),(0.26±0.11),(0.34±0.29),(0.74±0.59), and 0.25 h. The method is proved specific and repeatable and is suitable for the determination of psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, pso-ralen, isopsoralen, methylnissolin, and neobavaisoflavone in the rat plasma, which can be applied to pharmacokinetic study.

目的:建立补肺活血胶囊超高效液相色谱法(UPLC)指纹图谱.方法:采用ACQUITY UPLC BEH C18色谱柱(100 mm×2.1 mm,1.7μm),乙腈(A)-0.1％ 甲酸水溶液(B)为流动相梯度洗脱,流速为0.4 mL·min–1,检测波长为246 nm,建立补肺活血胶囊指纹图谱;采用UPLC串联四极杆飞行时间质谱法结合对照品对所建立的指纹图谱共有峰进行定性分析;采用主成分分析(PCA)进一步对影响补肺活血胶囊质量均一性的差异性成分进行筛选.结果:依据11批样品建立的指纹图谱相似度评价均大于0.969;指纹图谱中共标定20个共有峰,对其中18个共有峰进行了化学成分鉴定.PCA显示,影响补肺活血胶囊批间质量均一性的主要化学组分为补骨脂苷和异补骨脂苷.结论:所建立的UPLC指纹图谱方法专属性强,可用于补肺活血胶囊的质量评价.

Ethnopharmacological relevance: Guizhi-Fuling capsule (GZFL), a well-known herbal remedy, has been widely used to treat primary dysmenorrhea (PD). Hence, systematic identifying multiple active ingredients and the involved mechanism is essential and urgently needed for GZFL. Aim of the study: This study was planned to assess the pharmacokinetics of GZFL in rats, and identify whether these GZFL-derived absorbed components (ACs) contribute to the efficacy of source herbs and relevant mechanism. Materials and methods: The in vivo pharmacokinetic profile of 11 phytochemicals and 13 metabolites in healthy and PD rats were evaluated using liquid chromatography with mass spectrometry (LC-MS/MS). Whereafter, the introduced contribution strategy assessed ACs' effect (doses = their contents in GZFL) in PD rats with the mechanism. Result: The pharmacokinetic profiles of prototypes and metabolites differed in healthy and PD rats. As a main proxy of GZFL, 11ACs exerted an anti-PD effect (improvement of indexes for writhing latency, writhing time, PGF(2 alpha)/PGE(2), TXB2/6-keto-PGF(1 alpha) and beta-EP) by regulating PI3K-Akt/ERK pathway. Conclusion: As a paradigmatic example, 11ACs contributed an average of 113.55% to GZFL in terms of anti-PD efficacy, providing an approach to rapidly, accurately and consistently identify the bioactive components and their pathway from herbs.

目的:采用超高效液相色谱(UPLC)建立八珍益智方指纹图谱和多成分定量分析方法.方法:采用ACQUITY UPLC HSS T3色谱柱(100 mm×2.1 mm,1.8μm),流动相为乙腈(A)-0.2％甲酸水溶液(B),梯度洗脱,流速0.4 mL·min-1,检测波长254 nm(甘草酸)及300 nm(原儿茶酸、甘草苷、橙皮苷、异甘草苷、川陈皮素、桔皮素).通过对照品比对与Q-TOF/MS技术对共有峰进行指认,采用UPLC-PAD进行含量测定.结果:测得10批次八珍益智方样品的指纹图谱与对照指纹图谱的相似度均大于0.936;共确定八珍益智方指纹图谱19个共有峰,指认14个化学成分,对其中原儿茶酸、甘草苷、橙皮苷、异甘草苷、川陈皮素、桔皮素、甘草酸7个成分进行含量测定.上述7个成分在一定浓度范围内呈良好线性关系,相关系数均大于0.9999;方法学考察结果显示各项指标均符合要求,回收率范围为92.8％～106.7％,RSD小于4.2％.10批样品中原儿茶酸、甘草苷、橙皮苷、异甘草苷、川陈皮素、桔皮素、甘草酸含量范围分别为0.006～0.017、0.035～0.220、0.622 ～2.113、0.015 ～0.045、0.007～0.022、0.003～0.010、0.237～0.642 mg·g-1.结论:所建立的指纹图谱及多指标成分含量测定的分析方法稳定可行,专属性强,可用于八珍益智方的质量控制,为新药研发和临床安全用药提供依据.

目的 建立扎里奴思方的UPLC-PDA指纹图谱及多成分含量测定方法.方法 采用Waters ACQUITY UHPLC T3(2.1 mm×100 mm,1.7μm)色谱柱,流动相为乙腈(A)-0.1％甲酸水溶液(B),梯度洗脱,流速0.4 mL/min,柱温30℃,检测波长317 nm;采用UPLC-PDA对其中9种物质进行含量测定.结果 共确定扎里奴思方指纹图谱13个共有色谱峰,通过对照品比对出其中9个共有峰.10批样品指纹图谱相似度为0.923～0.983.9种物质在一定浓度范围内呈良好线性关系,r值均大于0.9990;含量测定结果表明肉桂酸、没食子酸含量较高.各物质回收率在98.82％～102.91％之间,RSD均小于2.52％;样品在24 h内稳定,重复性良好.结论 建立了扎里奴思方指纹图谱分析方法及含量测定方法,该方法简便、重复性良好,可为进一步完善扎里奴思方质量控制方法提供参考.